A strong effect of growth medium and organ type on the identification

Journal of Experimental Botany, Vol. 60, No. 5, pp. 1409–1425, 2009Perspectives on Plant Development Special Issuedoi:10.1093/jxb/erp084

RESEARCH PAPER

A strong effect of growth medium and organ type on theidentification of QTLs for phytate and mineral concentrationsin three Arabidopsis thaliana RIL populations

Artak Ghandilyan1, Nadine Ilk2, Corrie Hanhart1, Malick Mbengue1, Luis Barboza1, Henk Schat3,

Maarten Koornneef1,2, Mohamed El-Lithy1,4,*, Dick Vreugdenhil4, Matthieu Reymond2 and Mark G. M. Aarts1,†

1 Laboratory of Genetics, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands2 Max-Planck-Institute for Plant Breeding Research, Carl-von-Linne-Weg 10, D-50829 Koln, Germany3 Ecology and Physiology of Plants, Faculty of Biology, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands4 Laboratory of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

Received 4 February 2009; Revised 20 February 2009; Accepted 25 February 2009

Abstract

The regulation of mineral accumulation in plants is genetically complex, with several genetic loci involved in the

control of one mineral and loci affecting the accumulation of different minerals. To investigate the role of growth

medium and organ type on the genetics of mineral accumulation, two existing (Ler3Kond, Ler3An-1) and one new

(Ler3Eri-1) Arabidopsis thaliana Recombinant Inbred Line populations were raised on soil and hydroponics as

substrates. Seeds, roots, and/or rosettes were sampled for the determination of their Ca, Fe, K, Mg, Mn, P or Znconcentrations. For seeds only, the concentration of phytate (IP6), a strong chelator of seed minerals, was

determined. Correlations between minerals/IP6, populations, growth conditions, and organs were determined and

mineral/IP6 concentration data were used to identify quantitative trait loci (QTLs) for these traits. A striking

difference was found between QTLs identified for soil-grown versus hydroponics-grown populations and between

QTLs identified for different plant organs. Three common QTLs were identified for several populations, growth

conditions, and organs, one of which corresponded to the ERECTA locus, variation of which has a strong effect on

plant morphology.

Introduction

Plants generally obtain the minerals for their growth from

the media they live on. The (bio)-availability of essential

minerals depends on their solubility in the growth media and

on their binding strength to soil particles. Many minerals are

cationic metals, which are generally taken up as hydrated

ions and/or as metal–chelate complexes (Clemens et al.,2002). Factors like soil structure and pH affect the bio-

availability of minerals to plants. Mineral requirements and

the capacity to accumulate them are species-dependent.

Mineral uptake, translocation, and storage processes in

various tissues and cellular compartments are vital for the

plant and need to be maintained within appropriate physio-

logical limits (Clemens, 2001). Therefore, firm regulatory

mechanisms are in place to control mineral uptake at the

organ and cellular level. At the moment, little is known

about the genes controlling the variation within species for

cationic mineral uptake, distribution, phytate biosynthesis

and storage in plants (Maser et al., 2001; Raboy, 2003;

Ghandilyan et al., 2006). Identification of these genes willincrease our understanding of the mineral uptake and

distribution process and may facilitate the improvement of

plant nutrient content and use efficiency with potentially

beneficial effects on crop yield and quality.

Improving our knowledge about the genetic control of

plant mineral concentration and the concentration of anti-

nutrients can also contribute to improved human health.

* Present address: Menoufiya University, Botany Department, Faculty of Science, Shebin El-Kom, Menoufiya (province), Egypt.y To whom correspondence should be addressed: E-mail: [email protected]ª The Author [2009]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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Cereals, vegetables, and fruits, and the products made from

them, make up a large part of human nutrition, especially in

vegetarian or vegan diets. Nutritional deficiencies account

for almost two-thirds of childhood deaths worldwide

(Welch and Graham, 2004). The major cause of mineral

malnutrition found among humans is the predominant

consumption of plant-based foods that contain inadequate

levels of bioavailable minerals. The bioavailability of manycationic minerals from food for human consumption is

severely reduced by the presence of anti-nutrients in food,

which can form strong complexes with cationic minerals.

One of these anti-nutrients is phytate also known as inositol

hexakisphosphate (IP6). For plants, phytate is the major

source of phosphorus for germinating seeds and hence is

important for seedling vigour, but for cereal-derived food

products it is a major anti-nutrient.Arabidopsis thaliana (Arabidopsis) is a molecularly and

genetically well-characterized plant species, which is very

suitable for large-scale genetic analysis of mutants and

natural variants. Given the availability of well-genotyped

mapping populations, such as Recombinant Inbred Line

(RIL) populations, Quantitative Trait locus (QTL) analysis

is a powerful technique to study complex traits of the

genetic differences that are present within the speciesArabidopsis (Koornneef et al., 2004).

Particularly since RIL populations represent an ‘immor-

tal’ genetic resource (homozygous lines that can easily be

propagated after self-fertilization), many replicates of iden-

tical lines can easily be studied in many different environ-

ments, and thus investigate thoroughly the genetic

component of the environmental response.

The aim of the research presented here is to study the

genetic variation for the accumulation of minerals in seeds,

rosettes, and roots of Arabidopsis grown on different media.

It is expected that there is a genetic control of mineral

accumulation in Arabidopsis organs, and it is conceivable

that this control will be specific for the type of organ underinvestigation. In addition, it is also expected that this

genetic control depends on the substrate used for plant

cultivation, since mineral bioavailability can vary. In this

study, a situation of high minerals bioavailability (plants

raised on hydroponic medium) was compared with a situa-

tion more related to an agronomic scenario with reduced

mineral bioavailability (soil-grown plants).

Given that accessions differ in their genetic composition,the analysis of similar traits in different populations derived

from contrasting accessions enables us to sample the genetic

variation and basis of a specific trait within a species. Three

different RIL populations grown on different media were

studied. The soil medium was common for the three

populations, whereas the hydroponic system was used for

two populations that were also grown on soil. For each

growing scenario, accumulation was quantified for sevenminerals elements (Ca, Fe, K, Mg, Mn, P, and Zn) and for

phytate in different organs (seed, rosette, and root) and

QTLs for this set of traits were detected. Mapping QTLs

using the same populations under different conditions would

enable us to distinguish common QTLs which are involved in

Fig. 1. Frequency distributions of the mineral (Zn, Mn, Fe, K, Ca, and Mg; lmol g�1 DW) and phytate (IP6; mg g�1 DW) concentrations

in seeds of the Ler/Eri-1 RIL population grown on soil. Arrows indicate the levels in the parental lines, with the thick arrows indicating Ler

and the slim arrows indicating Eri-1.

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mineral homeostasis, whatever the growing scenario, and/or

QTLs which are organ- or environment-specific. Based on

previous data for mineral levels in seeds analysed in 21

accessions (Vreugdenhil et al., 2004), Landsberg erecta (Ler),

Kondara (Kond), and Antwerp-1 (An-1) accessions were

selected and the available RIL populations derived from the

inter-accession crosses Ler/Kon and Ler/An-1 (El-Lithy

et al., 2006) were analysed. In addition, a new mapping

population, derived from the cross between Ler and acces-

sion Eringsboda-1 (Eri-1) was generated.

Fig. 2. Frequency distributions of the mineral (Zn, Mn, Fe, K, Ca, and Mg; lmol g�1 DW) and phytate (IP6; mg g�1 DW) concentrations

in seeds of the Ler/Kond RIL population grown on soil (dark) and hydroponics (light). Arrows indicate the levels in the parental lines, with

the thick arrows indicating Ler and the slim arrows indicating Kond.

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Materials and methods

Plant material and growing conditions

Arabidopsis thaliana accessions Landsberg erecta (Ler, N20),

Eringsboda-1 (Eri-1, CS22548; collected in South Sweden),

Kondara (Kond, CS6175; collected in Tadjikistan),

and Antwerp (An-1, N944; collected in Belgium) and the

RIL populations Ler/Eri-1, Ler/Kond, and Ler/An-1, were

grown in the experiments described by El-Lithy et al. (2006).

The parents and populations were grown once on soil (in

a greenhouse) and once on hydroponics (in a climate

chamber). The Ler/Eri population was only grown on

soil. All populations were grown in the same greenhouse and

the same climate chamber under the same settings

Fig. 3. Frequency distributions of the mineral (Zn, Mn, Fe, K, Ca, Mg, and P; lmol g�1 DW) concentrations in seeds of the Ler/An-1 RIL

population grown on soil (dark) and hydroponics (light). Arrows indicate the levels in the parental lines, with the thick arrows indicating Ler

and slim arrows indicating An-1.


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(daylength, temperature, RH; see below), but not at the same

time.

For plants growing on soil, seeds were placed on demi-

water-soaked filter paper in 6 cm Petri dishes and kept for 4

d in the cold (4 �C) to break any residual seed dormancy

and to ensure uniform germination. Afterwards, the Petridishes were transferred to 24 �C in light for 1 d to initiate

germination. Germinated seedlings were placed on soil [a

peat:perlite (4:1 v/v) or a peat:sand (3:1 v/v) mixture], one

plant per 6 cm clay pot, six plants per genotype in two

replications. Replications were randomized in the plot using

a randomized two-block design to reduce environmental

effects. Plants grew in an air-conditioned greenhouse, with

70% relative humidity, supplemented with additional light(model SON-T plus 400 W, Philips, Eindhoven, The

Netherlands) providing long-day conditions (16 h light),

and maintained at a temperature of 22–25 �C during the day

and 18 �C at night. For plants growing on hydroponics,

seeds were grown on a standard hydroponics solution

suggested for Arabidopsis (Tocquin et al., 2003), in a phyto-

tron at 20 �C with a relative humidity of 70% and a light

intensity of 40 W m�2 for 12 h d�1. The hydroponics set-up

consisted of a 9.0 l tray (liquid medium container) covered

with a firm, non-transparent black plastic lid containing

nine rows of nine holes. Each hole received a 0.5 ml

microfuge tube, the tip of which was cut off. The tubeswere filled with 0.55% agar (weight/volume) prepared with

deionized water. Seeds were placed on the agar surface of

the microfuge tubes, one seed per tube to yield nine plants

per genotype in two replications. Replications were ran-

domized in the trays using a randomized two-block design

to reduce environmental effects.

For soil-grown plants of the Ler/Kond and Ler/An-1

populations, samples of ripe dry seeds were harvested fromeach of the two blocks for further analysis, each consisting

of the seeds coming from six plants. For the hydroponi-

cally-grown Ler/An-1 population, samples of ripe dry seeds

were harvested from each of the two blocks for further

analysis, each consisting of the seeds coming from eight

plants. The ninth plant of each line in each block was grown

Fig. 4. Frequency distributions of Zn, Mn, and Fe (lmol g�1 DW) concentrations in rosettes (dark) and roots (light) of the Ler/Kond RIL

population (above the dashed line) and in rosettes of the Ler/An-1 RIL population grown on hydroponics (below the dashed line). Arrows

indicate the levels in the parental lines, with the thick arrows indicating Ler and the slim arrows indicating Kond or An-1.


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for 3 weeks only and the full rosette of each plant was

harvested for further analysis. For the hydroponically-

grown Ler/Kond population, samples of ripe dry seeds were

harvested from each of the two blocks for further analysis,

each consisting of the seeds from the nine plants per line.

To obtain rosette material, the experiment was repeated,

but plants were only grown for 3 weeks, after which nine

rosettes and nine root systems per line were harvested forfurther analysis.

Phenotypic analysis

Tissue concentrations of Zn, Mn, Fe, K, Ca, and Mg were

measured using Atomic Absorption Spectrometry (AAS).

Two replicate samples per line were analysed for seed and

rosette minerals, one sample was analysed for root minerals.

Each sample consisted of approximately 100 mg oven-dried

and ground rosette material from nine plants, up to 60 mgoven-dried and ground root material from 18 plants, or 100

mg of seeds from the bulk harvest of six plants. Tissues

were put in a Teflon cylinder together with 2 ml acid-mix

(HNO3:HCl, 4:1 v/v), closed tightly and mineralized for 7 h

at 140 �C. After cooling, each digest was diluted with 3 ml

deionized water and transferred to a sterile 15 ml tube.

Different dilution samples were made, depending on the

expected concentration of each mineral before measuringthe minerals with an Atomic Absorption Spectrophotome-

ter (Perkin Elmer AAS 1100; Perkin Elmer, Rodgau-

Judesheim, Germany). For seeds of the Ler/An-1 popula-

tion, the total P concentrations were measured using

a spectrophotometric method described by Chen et al.,

(1956). For seeds of the Ler/Eri and Ler/Kond populations,

the phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate,

IP6) concentrations were measured, rather than total P, aspreviously described by Bentsink et al. (2003).

All Zn, Mn, Fe, K, Ca, Mg, and P mineral concentra-

tions are presented in lmol g�1 DW units, which is most

common in mineral analysis. These convert to lg g�1 DW

units, as follows: 1 lg g�1 is 65.4 lg g�1 for Zn, 54.9 lg g�1

for Mn, 55.8 lg g�1 for Fe, 39.1 lg g�1 for K, 40.1 lg g�1

for Ca, 24.3 lg g�1 for Mg, and 31 lg g�1 for P. The

phytate concentrations are presented in mg g�1 DW. 1 mgg�1 phytate (C6H12O24P6) corresponds to 654 mmol g�1.

Construction of the Ler/Eri mapping population andgenotyping

An F2 population derived from a cross between Ler

(maternal parent) and Eri-1 (paternal parent) (CS22548)

was propagated by single seed descent for nine successive

generations. 110 Recombinant Inbred Lines were obtained.

For genotyping, the flower buds of three F9 plants per line

were collected. DNA extraction used the Wizard� Magnetic96 DNA Plant System (Promega; www.promega.com)

according to the manufacturer’s instructions. Genomic

DNA was used for genotyping using AFLP and SSLP

markers. 90 AFLP markers were obtained using one primer

combination (E, EcoRI primer GACTGCGTACCAATTC

and M, MseI primer GATGAGTCCTGAGTAA). In

addition, a set of 39 SSLP markers distributed over the five

Arabidopsis chromosomes were used to genotype all the

lines (see Supplementary Table 1 at JXB online).

A genetic map has been created using JoinMap� 4

(www.kyazma.nl). All the genetic information from AFLP

and SSLP markers has been used. To avoid similarities of

loci due to a low frequency of recombination betweenmarkers within the population, 40 co-segregating AFLP

markers have been removed from the analysis. A total of 89

markers have then been used to build the genetic map. The

grouping was based upon the JoinMap�4 test for in-

dependence with the LOD score as the statistic. The

Kosambi function was used in a regression mapping

algorithm (Stam, 1993) to build the genetic map. The

known physical positions of the SSLP markers based onthe reference sequence of Arabidopsis thaliana (TAIR:

www.Arabidopsis.org) was used to assign each linkage

group to a specific chromosome.

Statistical tests and QTL mapping

For all statistical analyses the SPSS package version 15.0was used. Differences in mean trait values of the genotypes

were analysed by Univariate Analysis of Variance using the

Dunnett’s pairwise multiple comparison t tests in the

Table 1. Concentration ratios for minerals (Zn, Mn, Fe, K, Ca, Mg,

and P) and phytate (IP6) for three RIL populations (Ler/Kond, Ler/

An-1, and Ler/Eri-1) corresponding to different organs (seed,

rosette or root)

Seed concentrations are considered for plants grown on soil orhydroponics (hydrop). For ratios with one organ/growth conditioncombination, the value represents the highest average mineral orphytate concentration of any RIL, divided by the lowest averagemineral or phyate concentration of any RIL. The ratios shown forcomparisons of different organs or growth conditions represent themax/min ratio for the first organ/growth condition divided by themax/min ratio for the second organ/growth condition. Rosette androot values were only obtained for plants grown on hydroponics.

Ratio Ler/Kond Ler/An-1

Zn Mn Fe K Ca Mg IP6 Zn Mn Fe K Ca Mg P

Seed soil 1.6 1.9 2.8 1.8 1.8 1.4 2.6 1.5 1.8 3.0 2.1 1.8 1.4 1.4

Seed hydrop. 1.6 1.8 2.3 5.0 3.3 1.5 2.5 1.9 2.6 2.1 2.1 1.6 1.5 1.7

Rosette 1.9 1.8 2.7 2.7 1.9 2.7

Root 3.6 3.3 3.9

Seed soil/

hydrop.

1.0 1.1 1.2 0.4 0.5 0.9 1.0 0.8 0.7 1.4 1.0 1.1 0.9 0.8

Rosette/seed 1.2 1.0 1.2 1.4 0.7 1.3

Root/seed 2.3 1.8 1.7

Root/rosette 1.9 1.8 1.4

Ler/Eri-1

Zn Mn Fe K Ca Mg IP6

Seed soil 1.7 1.6 2.2 1.8 1.9 1.4 2.6


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www.promega.com

www.kyazma.nl

http://www.Arabidopsis.org

General Linear Model module of the package. For each

analysis, trait values were used as dependent variables and

genotypes were used as fixed factors. Tests were performed

2-sided with a significance threshold level of 0.05. The

independent samples t test of the package was used to

determine mean differences between two individual lines.

Correlation analyses were performed by calculating the

Pearson or Spearman correlation coefficients.QTL mapping was performed using the MapQTL�

software version 5.0 (www.kyazma.nl) and a complete pair-

wise search for conditional and co-adaptive epistatic inter-

actions for each trait was done (P <0.001, determined by

Monte Carlo simulations) using the EPISTAT Statistical

Package (Chase et al., 1997). In addition, interactions among

QTLs were analysed using co-factors (taken as the markers

closest to a QTL) as fixed factors and the traits as dependent

variables in a Univariate Analysis of Variance. Models

included marker main effects and interactions among them.

Results

To investigate the genetics of seed, rosette, and root mineral

and phytate homeostasis, immortal RIL populations were

Fig. 5. Average seed, rosette, and root mineral (Zn, Mn, Fe, K, Ca, Mg, and P) and phytate (IP6) concentrations (6SE) in the Ler/Kond,

Ler/An-1, and Ler/Eri-1 RIL populations grown on soil and on hydroponics. Except for the K concentrations in seeds, all differences

between mineral concentrations for different organs or conditions were significant (P <0.05).


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www.kyazma.nl

studied that were derived from the inter-accession crosses

Ler3Kond, Ler3An-1 (El-Lithy et al., 2006), and Ler3Eri-1

(a new population), which were grown on soil and, for two

populations, that were also grown in a hydroponics system.

Variation in plant mineral and phytate concentrations

The traits studied, which are the Ca, Fe, K, Mg, Mn, P, Zn,

and phytate concentrations of seeds, rosettes, and roots,

demonstrated large segregations in all three RIL populations

grown in both soil and hydroponic conditions (Figs 1, 2, 3,

4). For several traits, such as seed Zn and K concentrations

in Ler/Kond RILs, the segregations were transgressive,

meaning that values for the RILs were substantially largerand/or smaller than both of the parents, whereas for some

others, such as rosette Zn concentrations in Ler/Kond RILs,

the RIL values were intermediate to the parental values.

Mineral concentrations varied 1.4–3.0-fold for seeds of the

three soil-grown populations and 1.5–5.0-fold for seeds of the

two hydroponics-grown populations. For the rosettes of

hydroponically grown plants, mineral concentrations varied

1.8–2.7-fold in both populations. The root mineral concen-trations of the Ler/Kond RIL population grown on hydro-

ponics varied 3.3–3.9-fold (Figs 1, 2, 3, 4; Table 1). In

general, the concentration ranges were comparable for most

minerals when comparing populations and conditions. Only

for seed K and Ca concentrations for hydroponically grown

plants, was the variation in the Ler/Kond population about

twice as large as in the Ler/An-1 population and also as in

the soil-grown Ler/Kond population. The levels of seedmineral concentrations also depended on the growth con-

ditions (Fig. 5). For both populations grown on soil and on

hydroponics, the maximum difference between concentra-

tions was 3.4-fold. In particular, seed K concentrations were

similar for both conditions. For the Ler/Kond population the

seed mineral concentrations were higher when the RILs were

grown on soil than on hydroponics, except for Fe and K.

For Fe, the seed concentrations were higher in the hydro-ponically grown population. For the Ler/An-1 population

the seed Zn, Mn, Fe, and Mg concentrations were higher

when the RILs were grown on soil, and the seed Ca and P

concentrations were higher for the hydroponically grown

plants. In general, the Zn and Fe concentrations in the roots

were higher than in rosettes and seeds (Fig. 5). The seed Fe

concentrations were higher than rosette Fe concentrations in

the Ler/Kond population, whereas it was the reverse for the

Ler/An-1 population.

All these results indicate that, in general, mineral and

phytate concentrations and their variation levels depended on

the sampled organ, population, and/or growing conditions.

Relationship between the traits

Both negative and positive correlations were observed

between traits (Tables 2, 3). The correlation of the seed Zn

concentration with the seed Mn, Fe, K, Mg and P concen-

trations in the Ler/An-1 RILs, was observed in the popula-

tion grown on both soil and hydroponics. Root Zn and Fe

concentrations (Ler/Kond) were always negatively correlated

and no correlations were observed between the seed and

rosette Zn and Fe concentrations in the Ler/Kond RILsgrown on hydroponics. Many of the correlations observed

within a population, like the seed Zn and Fe concentrations

in the Ler/Kond RILs, were not stable over the two different

environments, but only observed in one condition (in this

case when the population was grown on soil). Seed P and

Mn concentrations were positively correlated with all the

other seed mineral concentrations in Ler/An-1 RILs grown

on soil, whereas some of the correlations for soil-grownplants were not significant when the population was grown

on hydroponics. Also the reverse can be observed, such as

for the seed IP6 and Zn concentrations, which were

correlated in the Ler/Kond RIL population only when grown

on hydroponics. Where often positive correlations were

found between mineral concentrations in seeds of both soil-

grown populations, they were often negative between soil

and hydroponics-grown plants of the Ler/Kond population.In general, correlations of Zn concentrations with other

mineral concentrations were different from those between

concentrations of Fe and the other minerals.

Overall, it was observed that (i) the concentrations of

a mineral in the same organ generally did not correlate

between the two growth conditions; (ii) the concentrations

of different minerals, measured in the same organs, grown

under the same conditions generally did not correlate; and(iii) the concentrations of a mineral when measured in

different organs (whether or not grown under the same

conditions) generally did not correlate. Also, even if

correlations were found to be statistically significant, the

correlation was often not very high, seed mineral concen-

trations of soil-grown plants being an exception. This

general absence of robust correlations between mineral

concentrations over all conditions, organs, and populationssuggests that there are many condition-, organ-, and

population-specific (genetic) factors to control mineral

concentrations.

Genetic map of the Ler/Eri-1 population

This report describes a new RIL population, made from

a cross between Ler and Eri-1. The genetic map obtained

Table 2. Correlation coefficients (r) of mineral (Zn, Mn, Fe, K, Ca,

and Mg) and phytate (IP6) concentrations in seeds of the Ler/Eri-1

RIL population grown on soil

Significant negative correlation coefficients are highlighted in bold.Significance threshold levels: * P <0.05; ** P <0.01; *** P <0.001.

Mn Fe K Ca Mg IP6

Zn 0.48*** 0.58*** 0.06 0.01 0.24** 0.05

Mn 0.20* 0.10 0.33*** 0.23* 0.33***

Fe –0.04 –0.11 0.12 –0.20*

K –0.20* 0.41*** 0.38***

Ca 0.20* 0.24*

Mg 0.27**


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Table 3. Correlation coefficients (r) of mineral (Zn, Mn, Fe, K, Ca, Mg, and P) and phytate (IP6) concentrations in seeds, rosettes, and roots of the Ler/Kond (upper right diagonal

half) and in seeds and rosettes of the Ler/An-1 RILs (bottom left diagonal half) grown on soil and hydroponics

Negative significant correlation r values are highlighted (grey). Significance threshold levels: * P <0.05;** P <0.01;*** P <0.001.

Seed (soil) Seed (hydrop) Rosette Root

Zn Mn Fe K Ca Mg IP6or Pa

Zn Mn Fe K Ca Mg IP6or Pa

Zn Mn Fe Zn Mn Fe

Seed

(soil)

Zn 0.30** 0.49*** –0.06 0.09 0.12 –0.04 0.09 –0.04 –0.01 0.06 –0.16 –0.02 0.13 0.01 0.09 0.04 –0.03 –0.04 0.04

Mn 0.34** 0.02 0.12 0.57*** 0.23* 0.32*** –0.27** 0.28** –0.13 0.02 0.06 0.23 0.26* –0.05 0.08 0.18 –0.15 0.02 0.13

Fe 0.53*** 0.34*** –0.20* –0.03 0.14 –0.15 0.18 –0.23* 0.11 –0.01 –0.29** 0.10 0.05 0.04 –0.07 0.11 0.01 –0.12 0.04

K 0.26* 0.31** 0.09 –0.16 0.49*** 0.33*** –0.14 0.11 –0.35*** 0.24* –0.16 –0.09 0.11 –0.02 0.12 0.24* –0.14 0.17 0.07

Ca 0.17 0.41*** 0.23* –0.30** –0.09 0.16 –0.14 0.34*** 0.03 0.12 0.33*** 0.06 –0.12 0.25** 0.12 0.09 –0.07 –0.05 0.02

Mg 0.40*** 0.40*** 0.33*** 0.55*** –0.08 0.37*** –0.16 0.06 –0.30** 0.28** –0.51*** 0.23 0.37*** –0.43*** 0.07 0.24* –0.15 –0.01 0.25**

IP6 or Pa 0.34** 0.56*** 0.24* 0.36*** 0.43*** 0.60*** –0.38*** 0.09 –0.32** 0.01 –0.09 0.15 0.24* –0.07 0.21* 0.09 –0.30** –0.09 0.18

Seed

(hydrop)

Zn 0.08 0.16 0.25* 0.30** 0.03 0.23* 0.09 –0.01 0.16 0.07 –0.11 0.04 –0.21* 0.10 0.08 –0.19* 0.15 0.09 –0.10

Mn 0.08 0.21* 0.22* 0.35*** –0.04 0.29** 0.07 0.30** 0.19* –0.03 0.30*** 0.13 0.02 0.01 0.20* 0.00 0.13 0.09 –0.01

Fe 0.01 –0.03 0.40*** 0.04 –0.07 0.04 –0.24* 0.37*** 0.51*** –0.46*** 0.24* 0.23* –0.18 0.03 0.02 –0.03 0.11 0.02 –0.08

K 0.19 0.24* 0.11 0.30** 0.10 0.24* 0.40*** 0.42*** –0.20* –0.12 –0.43*** –0.16 0.15 –0.02 0.11 –0.07 –0.03 –0.03 –0.13

Ca –0.15 –0.07 –0.07 –0.12 0.07 –0.05 –0.16 –0.11 0.32** 0.30** –0.63*** –0.07 –0.34*** 0.38*** 0.04 –0.20* 0.07 0.11 –0.12

Mg 0.12 0.22* 0.22* 0.16 –0.01 0.32*** 0.34*** 0.47*** 0.05 0.10 0.60*** –0.27** 0.06 –0.13 –0.05 0.02 0.11 –0.13 –0.12

IP6 or Pa 0.12 0.24* 0.07 0.23* 0.17 0.27** 0.42*** 0.56*** –0.10 –0.13 0.72*** –0.39*** 0.78*** –0.34*** 0.10 0.25** –0.05 –0.24* 0.06

Rosette Zn –0.11 0.02 –0.11 0.16 –0.18 –0.09 –0.17 0.17 0.07 0.07 0.00 –0.08 –0.11 –0.06 0.41*** 0.08 0.00 0.10 –0.15

Mn 0.24* 0.11 0.26** 0.24* –0.05 0.25** 0.06 0.31*** 0.22* 0.24** 0.24** –0.18 0.33*** 0.23* 0.21* 0.28** –0.24** 0.20* –0.05

Fe 0.00 –0.03 –0.04 0.11 –0.15 –0.06 –0.27** 0.22* 0.18 0.08 –0.03 –0.10 –0.02 –0.07 0.23* 0.27** –0.18* 0.03 0.23*

Root Zn –0.02 –0.20*

Mn 0.01

a Total P concentrations for Ler/An-1 and IP6 concentrations for Ler/Kond RILs.

Arabidopsis

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lgenetic

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for this Ler/Eri-1 RIL population has a length of 365 cM

(Fig. 6), which is in the same range as Arabidopsis genetic

maps obtained from other, different crosses (Alonso-Blanco

et al., 1998; Loudet et al., 2002; El Lithy et al., 2006). The

markers are distributed along the five chromosomes with

a genetic distance between two successive markers of 4 cM,

on average, with a maximum of 12 cM (between markers

T2N18 and F17A22 on chromosome 2; between markersDF.76L and BH.120L-Col on chromosome 3; between

markers M4-36 and G3883 on chromosome 4; Fig. 6). No

significant allelic distortion has been observed for this new

mapping population.

Genetic analyses of mineral concentrations

To estimate the proportion of phenotypic variation in

a population that can be attributed to genetic variation, thebroad-sense heritability values for all traits (Fig. 7) were

calculated. The values varied between 10.6%, for seed Fe

concentrations in the Ler/Eri-1 population and 89.2% for

seed P concentrations in the Ler/An-1 population. Herit-

abilities for the mineral concentrations were higher in the

Ler/An-1 population, which is most likely a population

effect, as it is seen for both soil and hydroponically grown

plants, although differences in the growing conditionscannot be ruled out, since populations were tested at

different times. To identify the genetic factors responsible

for the mineral concentrations in roots, rosettes, and seeds,

a QTL analysis was performed. QTLs were identified for

each trait in at least one of the three populations tested

(Figs 6, 8; Tables 4, 5, 6). The total phenotypic variances

explained by the identified QTLs were over 50% of the

heritability values for most of the traits (Fig. 7). No major

QTL was identified for seed Zn and Fe concentrations inthe Ler/Eri-1 population, which was in line with the low

heritability values of these traits in this population. The

trait variation was not a good indicator for the number of

QTLs that could be identified or the total explained

variances for a trait. For instance, the fold difference

between RILs for seed Mg concentrations (1.5-fold) in the

Ler/Kond population grown on soil was one of the lowest

in comparison to other traits, but the total trait varianceexplained by QTLs was the highest (53.5). Also for seed K

concentration, the ranges observed among the Ler/Kond

RILs were twice as high when compared to the Ler/An-1

RILs. However, the heritability values, the number of

identified QTLs, and their total explained phenotypic

variance, were similar.

Many of the QTLs identified for the same trait in the

three populations co-located. However, the total number ofQTLs detected in the Ler/An-1 populations was much

larger than for the other two populations. Four hotspots

Fig. 6. Genetic map of the Ler/Eri-1 RIL population with QTLs identified for seed mineral (Zn, Mn, Fe, K, Ca, and Mg) concentrations of

plants grown on soil. QTLs are indicated on the right of the chromosomes with thin lines indicating 1-LOD intervals and the thick bars

indicating 2-LOD interval for each QTL.


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were found for co-locating QTLs: the region of chromo-

some 2 around the ERECTA gene; the top of chromosome

3, around marker NGA172; and two regions on chromo-

some 5, respectively, around markers SNP236 and MBK5.

The co-locations identified were not specific to macro-elements (e.g. K and Ca) or micro-elements (e.g. Zn and

Fe), but often involved both groups of elements.

Epistatic interactions among many loci were detected in

many cases, although most interactions did not explain

a major part of the phenotypic variance compared to the

main effects (Table 7). Several QTL positions for the

populations grown on soil were previously identified for the

same traits in the Ler/Cvi population (Bentsink et al.,2003;Vreugdenhil et al., 2004). These include K and Ca

QTLs on chromosome 1 and the Zn and Mn QTLs around

ERECTA. In addition, the QTLs located at the top of

chromosome 3, including the strong IP6 and P QTLs that

were found in all populations and which co-located with K,

Zn, and Mn QTLs at least in some of the studied

populations. The cluster of QTLs at the MBK5 marker (at

the bottom of chromosome 5), was only detected in the Ler/

Kond and Ler/Eri-1 populations.

Despite many QTL co-localizations when comparing

different populations grown under the same condition, only

a few QTLs co-localized when comparing the same pop-ulation grown under two conditions (hydroponics or soil).

This again illustrates that there is considerable difference

between the genetic control of mineral accumulation when

plants are grown on hydroponic medium or on soil. It also

illustrates the large effect of mineral bioavailability, pH or

other factors like root architecture on mineral concentration

occurring between the growing conditions. Besides the main

effect of the environment (growth condition) on the traits,many interactions were also detected between loci and

environment (growth condition) (Table 8). Significant

interactions with growth conditions were detected for all

the seed mineral concentrations (including P), in particular,

at the Erecta marker (Fig. 9). The mineral concentrations

varied depending on the allele at the Erecta marker (Ler,

Kond, or An-1); depending on the growth condition

(hydroponics or soil); and depending on the interactionbetween the locus and the growth condition (Genotype3

Environment interaction). For example, on hydroponics,

seed Fe concentrations were highest in plants carrying the

Kond allele at the Erecta marker, compared to plants

carrying the Ler or An-1 alleles. On soil, however, the seed

Fe concentrations were lowest in plants carrying the Kond

allele at the Erecta marker, while the lines carrying the Ler

and An-1 alleles showed comparable differences betweenseed Fe concentrations as on hydroponics. Thus, for Fe

concentration there is a clear G3E interaction. G3E

interactions at the Erecta marker were not found for seed

IP6 concentrations.

Discussion

Micronutrients, such as iron, zinc, vitamin A, and iodine,

are required by humans in small amounts only, but are

essential for good health. Women and children in Sub-

Saharan Africa, South and South-East Asia, Latin Amer-

ica, and the Caribbean are especially at risk of disease,

premature death, and impaired cognitive abilities becauseof diets lacking essential micronutrients (http://www.har-

vestplus.org/). In order to allow breeding for varieties with

a higher mineral content (bio-fortification) or a more

efficient uptake of minerals from the soil, knowledge on

the genetic variation of cationic mineral homeostasis and

the genes underlying allelic variation needs to be ex-

panded. In the past, little attention has been paid to

breeding for enhanced mineral content. With the HarvestPlus initiative that has changed. Recent efforts to breed for

micronutrient-enhanced rice grains showed that, although

there appears to be sufficient genetic variation for micro-

nutrient content in rice, it is a difficult trait to breed for

(Gregorio et al., 2008). The main difficulties are the

Fig. 7. Heritabilities and explained phenotypic variances (Total

Explained Variance) for the mineral (Zn, Mn, Fe, K, Ca, and Mg)

and phytate (IP6) concentrations in the Ler/Kond, Ler/An-1, and

Ler/Eri-1 RIL populations. Data are provided for seed mineral

concentrations of soil and hydroponically (hydrop) grown plants and

for rosette mineral concentrations of hydroponically grown plants.


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http://www.harvestplus.org/

http://www.harvestplus.org/

complexity of the traits, low heritabilities, and the high

environmental influence. Therefore we set about to in-

vestigate this in the model plant species Arabidopsis

thaliana.

Studying mineral homeostasis in plants grown on different

media will help to understand the differences in regulation

due to growth conditions, whereas studying mineral homeo-

stasis in different plant organs will help to understand thedifferences in the regulation due to organ specificity. To

study the genetics behind mineral and phytate homeostasis in

plants, segregating Arabidopsis populations were used based

on crosses of accessions Landsberg erecta (Ler) with

Kondara (Kond) and Antwerp (An-1) (El-Lithy et al.,

2006), for which it was known that the parents differed

significantly in seed mineral concentrations (Vreugdenhil

et al., 2004). In addition, a new mapping population wasincluded in this study, which is derived from a cross

between accessions Ler and Eringsboda. In addition to the

previously used accession Cape Verde Islands (Cvi; Vreug-

denhil et al., 2004), these four accessions cover a wide range

of ecological niches and should provide a good representa-

tion of available genetic variation for mineral concentra-

tions in Arabidopsis.

Variations in mineral concentrations of plants will de-pend on variations in many parameters, like mineral

mobilization, uptake, trafficking, and sequestration, which

are all relevant processes in the mineral transport pathway

from roots to shoots (Clemens, 2001). Roots excrete

compounds to acidify the environment and, in roots and

shoots, ligands and chelates are present to bind minerals.

These processes depend on the substrate on which the plant

is grown. The two growth media used here for cultivating

the RIL populations, soil and hydroponics, are expected to

differ in terms of mineral (bio)-availability, buffering, and

ion exchange capacities, with more variation for soilconditions than for hydroponics and, especially for hydro-

ponics, a generally higher bio-availability. Thus, in the

latter medium, the intention was to remove one of the

‘bottlenecks’ limiting plant mineral acquisition and accumu-

lation. Although none of the growth conditions led to

obvious mineral deficiency or excess symptoms, it is still

possible that there were fewer or more (bio)-available

minerals present in one substrate ompared with the other,which caused differences in plant mineral concentrations,

although both growth conditions were considered as

optimal. It is realized that the different bio-availabilities of

the various minerals in the growth substrates might also

cause differences in competition between the minerals for

mobilization, uptake, translocation, and sequestration lev-

els, considering the presence of the large proportion of

mineral high- and low-affinity transporters and chelatesshared among minerals (Maser et al., 2001;Clemens et al.,

2002). Our hypothesis was that QTLs identified for the soil-

grown populations, but not in the hydroponics populations,

Fig. 8. Genetic map of the Ler/Kond and Ler/An-1 RIL populations with QTLs identified for seed (s) mineral (Zn, Mn, Fe, K, Ca, and Mg)

and phytate (IP6) concentrations of plants grown on soil (in black) or hydroponics (in blue) and for rosettes (rs; in green netted) and roots

(rt; in brown striped) of hydroponically grown plants. QTLs are indicated on the left of the chromosomes for Ler/Kond and on the right of

the chromosomes for Ler/An-1 with the thick bars indicating 1-LOD intervals and thin lines indicating 2-LOD interval for each QTL.


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would correspond to loci involved in enhancing mineralbio-availability.

Variation in mineral concentrations was observed between

different organs when comparing RILs of one population.

This suggests that mineral concentrations are maintained in

plants in an organ specific manner. The rosette concentra-

tions were determined for plants which had not yet bolted.

Potentially, minerals accumulated in rosette leaves could be

available for later loading into the inflorescence and,eventually, seeds, although Waters and Grusak (2008a)

recently showed that seed loading in particular does not

depend on remobilization but rather on direct uptake by

roots from soil. It is not clear if this also holds for

mobilization to developing inflorescences. Considerable

transgression of phenotypic values in both directions for seed

mineral concentrations were detected in specific RILs, as was

also previously observed in Ler/Cvi RILs (Vreugdenhil et al.,

2004), suggesting that both accessions carry genes with allelescontributing to an increased or a decreased content of all

minerals tested.

Both negative and positive correlations were observed

among traits. Most robust are the correlations between Zn,

Fe, and Mn concentrations, which are largely independent

of the organ, population, or environment. These three

minerals are known to share components of their respective

mechanism for uptake from soil, transport into and out ofcells and cell organelles, and for chelation during vascular

transport (Maser et al., 2001), which could account for such

correlations. However, whereas most correlations for these

minerals in seeds of soil-grown plants are positive (suggest-

ing co-transport and co-chelation), when comparing other

organs, negative correlations are also found, suggesting

a limited availability of transport proteins or chelator

molecules causing competition between minerals. Also IP6

Table 4. QTLs affecting mineral (Ca, Fe, K, Mg, Mn, P, and Zn) and phytate (IP6) concentrations identified in the Ler/Kond RIL

population

Seed QTLs are determined for the population when grown on soil or hydroponics as indicated in brackets. Rosette and root QTLs are onlydetermined for the hydroponically grown population. For each QTL the chromosome number is indicated (Chrom), the genetic position (in cM),the closest genetic marker (Locus), the additive logarithm of odds value (LOD), the percentage of explained phenotypic variance (% Expl. var.)and the parental allele that increases the trait value (Effect).

Trait Chrom Position Locus LOD % Expl. var. Effect

Seed (soil) Mn 3 0 nga172 3.60 12.9 Kond

Mn 5 10.4 SGCSNP77 2.57 9.1 Ler

Fe 3 49.8 SGCSNP188 3.20 10.7 Ler

Fe 4 4.7 msat4-8 5.84 20.6 Kond

K 1 0 SGCSNP5 4.02 12.8 Ler

K 3 0 nga172 2.80 8.6 Kond

K 5 79.1 MBK5 3.95 12.5 Ler

Ca 5 10.4 SGCSNP77 6.25 23.5 Ler

Mg 1 24.2 SGCSNP251 3.97 8.7 Kond

Mg 4 4.1 FRI 4.40 9.6 Kond

Mg 4 39.1 SGCSNP152 5.09 11.5 Kond

Mg 5 0 SGCSNP93 2.72 5.7 Kond

Mg 5 79.1 MBK5 4.61 10.0 Ler

IP6 3 0 nga172 13.46 43.6 Kond

Seed (hydroponics) Zn 1 94.1 SGCSNP142 2.48 9.8 Kond

Zn 3 0 nga172 3.06 12.2 Ler

Mn 5 27.4 SGCSNP236 5.07 19.7 Ler

Fe 2 36.5 SGCSNP233 6.27 24.1 Kond

K 2 21.5 SGCSNP203 5.39 14.7 Ler

K 3 56.3 SGCSNP197 4.75 13.1 Ler

K 4 27.8 SGCSNP408 4.19 11.5 Kond

K 5 31.6 SGCSNP193 5.40 15.1 Ler

Ca 4 4.1 FRI 8.57 28.2 Ler

Ca 4 55.5 SGCSNP53 4.40 15.8 Ler

Ca 5 79.1 MBK5 4.91 12.4 Kond

Rosettes Zn 1 0 SGCSNP5 2.60 7.5 Ler

Zn 3 42.4 SGCSNP248 3.74 10.7 Ler

Zn 4 39.1 SGCSNP152 5.61 16.7 Ler

Mn 5 43.8 AthS0191 3.95 15.0 Ler

Mn 5 72.0 SGCSNP101 3.83 14.4 Ler

Fe 5 73.8 SGCSNP304 4.67 17.8 Ler

Roots Zn 1 68.9 nga128 2.80 10.9 Ler

Fe 3 2.8 SGCSNP114 3.94 14.5 Kond


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concentrations in seeds are frequently found to be corre-

lated with other minerals, particularly Zn, K, and Mg, inline with the physical co-locations of IP6 and these minerals

in different parts of Arabidopsis developing seeds (Otegui

et al., 2002).A genetic basis for mineral concentrations in the popula-

tions was founded by identifying many QTLs in each

population, often co-locating between the RIL populations.

For example, in all populations studied, the presence of

a Ler allele on the top of chromosome 3 resulted in lower

seed P/IP6 concentrations and, consequently, in QTLs.

These results suggest that the Ler allele at this locus differs

from the alleles in the other parental accessions. The samewas previously found by Waters and Grusak (2008b).

Regarding our initial hypothesis that QTLs identified for

both soil and hydroponics would represent loci not involved

in mineral bio-availability, this only holds for three loci at

which several soil and hydroponics seed mineral QTLs co-

localize. These three mineral QTL hotspots are associated

with the markers Erecta (chromosome 2), NGA172 (chro-

mosome 3), and MBK5 (chromosome 5). These three lociwere also hotspots for QTLs of life history traits found in

the Ler/Cvi population (Alonso-Blanco et al., 1999;Ungerer

et al., 2002). Co-localization of the QTLs for different traits

suggests a single pleiotropic locus to be involved in the

homeostasis of multiple traits. A straightforward explana-

tion for this co-localization would be if such a locus

represents a gene for which variation has a striking effect

on plant morphology or development, as that will verylikely also affect mineral homeostasis. For the Erecta locus

such an effect can easily be envisioned. The Ler parent,

which was used for all three populations, as well as the Ler/

Cvi population, carries a mutant allele of the ERECTA

gene. The ERECTA gene encodes a receptor protein kinase

protein, mutation of which has a drastic effect on plant

morphology (Torii et al., 1996). Previously the Erecta locus

has been identified as a major QTL for various traits(including mineral concentrations; Waters and Grusak,

2008b) in populations with Ler as one of the parents

(Llorente et al., 2005; Masle et al., 2005; Tisne et al., 2008).

The ERECTA gene, which was previously also shown to

affect seed yield-associated factors like plant total seed

number in Arabidopsis (Alonso-Blanco et al., 1999), was

Table 5. QTLs affecting mineral (Ca, Fe, K, Mg, Mn, P, and Zn)

and phytate (IP6) concentrations identified in the Ler/An-1 RIL

population

Seed QTLs are determined for the population when grown on soil orhydroponics as indicated in brackets. Rosette QTLs are onlydetermined for the hydroponically grown population. For each QTLthe chromosome number is indicated (Chrom), the genetic position(in cM), the closest genetic marker (Locus), the additive logarithm ofodds value (LOD), the percentage of explained phenotypic variance(% Expl. var.) and the parental allele that increases the trait value(Effect).

Trait Chrom Position Locus LOD % Expl.var.

Effect

Seed (soil) Zn 2 36.6 nga1126 11.06 36.8 Ler

Zn 3 29 SNP35 5.33 15.1 An-1

Zn 5 28.4 SNP236 2.86 7.6 Ler

Mn 2 34.8 Erecta 12.29 37.2 Ler

Mn 4 34.4 SNP295 2.98 7.4 An-1

Fe 2 32.8 SNP203 3.22 10.9 Ler

Fe 3 0 SNP105 5.39 19.6 An-1

K 1 34.3 SNP373 3.24 9.3 Ler

K 3 0 SNP105 3.45 9.8 An-1

K 5 30 nga139 7.98 24.8 Ler

Ca 1 61.3 nga128 8.22 25.5 Ler

Ca 3 24.1 SNP81 6.30 19.0 An-1

Mg 1 76.4 SNP177 3.02 7.2 An-1

Mg 2 34.8 Erecta 4.14 9.9 Ler

Mg 3 0 SNP105 7.99 21.0 An-1

Mg 5 18.6 SNP358 6.34 16.0 Ler

P 2 34.8 Erecta 5.12 8.4 Ler

P 3 0 SNP105 23.28 56.6 An-1

P 4 37.6 SNP199 2.96 4.9 An-1

Seed

(hydroponics)

Zn 2 36.6 nga1126 5.86 19.0 Ler

Zn 4 46 SNP334 3.52 10.9 Ler

Mn 1 15 SNP132 2.71 7.2 Ler

Mn 4 55.2 SNP232 2.93 7.8 Ler

Mn 5 28.4 SNP236 9.93 30.5 Ler

Fe 1 32 SO392 2.47 5.9 Ler

Fe 2 36.6 nga1126 4.31 10.8 Ler

Fe 3 0 SNP105 9.04 25.1 Ler

K 2 34.8 Erecta 5.25 13.5 Ler

K 3 0 SNP105 2.38 5.8 An-1

K 5 23.3 SNP130 3.38 9.6 An-1

K 5 79.7 SNP304 2.61 7.3 Ler

Mg 2 34.8 Erecta 4.19 12.4 Ler

Mg 5 28.4 SNP236 2.82 8.1 An-1

Mg 5 84.6 MBK5 3.35 9.7 Ler

P 2 17.8 F12A24b 3.42 6.3 Ler

P 3 0 SNP105 7.31 14.8 An-1

P 3 72.2 SNP188 2.77 5.1 Ler

P 5 28.4 SNP236 8.62 17.9 An-1

P 5 81.3 CIW10 4.59 8.7 Ler

Rosettes Zn 3 7.1 SNP114 2.74 8.5 Ler

Zn 4 48.8 SNP152 6.77 22.5 Ler

Mn 1 15 SNP132 4.03 10.8 Ler

Mn 4 55.7 F8D20 4.03 10.6 Ler

Mn 5 84.6 MBK5 2.99 7.7 Ler

Table 6. QTLs affecting seed mineral (Ca, Fe, K, Mg, Mn, P, and

Zn) and phytate (IP6) concentrations identified in the Ler/Eri-1 RIL

population

QTLs are determined for the population when grown on soil. Foreach QTL the chromosome number is indicated (Chrom.), thegenetic position (in cM), the closest genetic marker (Locus), theadditive logarithm of odds value (LOD), the percentage of explainedphenotypic variance (% Expl. var.) and the parental allele thatincreases the trait value (Effect).

Trait Chrom. Position Locus LOD % Expl. var. Effect

Mn 3 3.1 F22F7 2.48 9.8 Eri

K 2 29.7 M2-17 2.96 5.6 Eri

K 3 0.0 DF.252L 13.43 32.0 Eri

K 4 56.7 M4-33 5.14 10.1 Eri

K 5 45.5 DF.460E 3.01 5.7 Eri

IP6 3 0.0 DF.252L 9.25 31.9 Eri


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similarly located in the QTL interval for Zn and Mnconcentrations in plants identified in the Ler/Cvi population

(Vreugdenhil et al., 2004).

Co-localization of QTLs did not always reflect the

correlations observed between the traits. For instance,

although the correlation coefficient between seed IP6 and

Mg concentrations in Ler/Kond RILs grown on soil was

higher than between IP6 and Mn and K concentrations, the

identified QTL for seed IP6, Mn, and K concentrations didco-locate, whereas the QTL for seed Mg concentrations did

not. This is most likely due to the presence of other QTLs

for these minerals. Often the same QTLs were not found in

these hotspot clusters. This can be explained by the fact that

a QTL close to the border of significance can appear in one

but might not appear in another population. However, the

identification of generally different QTLs for mineral

accumulation in the same populations, when grown on soil

Table 7. Epistatic interactions (P <0.005) between two loci in the

LerKond, Ler/An-1, and Ler/Eri-1 populations

Traits are mineral (Ca, Fe, K, Mg, Mn, P, and Zn) or phytate (IP6)concentrations, with (h) or (s) indicating, respectively, seed traits ofhydroponics and soil-grown plants and (rs) indicating rosette mineraltraits. For each interaction the additive P-value of the interaction isindicated. Only statistically significant (P <0.05) interactions areshown. The percentage of phenotypic variance that is explained bythe interaction is indicated (% Expl. var.).

Trait Locus1

Locus2

AdditiveP-value

%Expl.var.

Ler/

Kond

Fe(h) SNP101 SNP295 0.0005 3.6%

Fe(rs) nga128 SNP104 0.0014 7.0%

Fe(s) nga6 SNP203 0.0008 6.0%

IP6(s) MBK5 SNP388 0.0005 5.6%

K(h) SNP114 SNP395 0.0001 8.1%

K(h) SNP135 SNP388 0.0009 8.2%

Mg(h) F12A24b SNP93 0.001 4.8%

Mg(h) SNP166 SNP93 0.0023 6.2%

Mn(h) msat4-3 SNP251 0.0024 3.9%

Ler/An-1 Ca(h) nga139 SNP220 0.0003 7.7%

Ca(s) SNP177 SNP35 0.0013 24.9%

Ca(s) SNP157 SNP204 0.0029 21.5%

Ca(s) SNP136 SNP304 0.0003 3.4%

Fe(s) SNP301 M4-41 0.0007 6.3%

Mg(s) SNP81 F6D8-94 0.0021 13.5%

Mn(rs) T27K12 SNP169 0.0003 8.2%

Mn(s) SO392 SNP184 0.0002 5.2%

Mn(s) F12A24b SNP373 0.0005 12.4%

Mn(s) M2-17 M2-5 0.0083 31.7%

P(h) SNP268 SNP295 0.0004 7.9%

P(h) M4-41 SNP77 0.0012 3.1%

Zn(h) F12A24b SNP295 0.0009 5.1%

Zn(rs) nga6 SNP236 0.0016 3.4%

Zn(rs) SNP114 CIW7 0.0002 6.0%

Zn(s) SNP391 CIW7 0.0006 15.0%

Ler/Eri-1 Zn(s) SO191 T6A23 0.0002 1.6%

Zn(s) NGA106 NGA129 0.0001 1.2%

Mn(s) BH.354E DF.119L 0.0008 5.3%

Mn(s) CH.318E NGA106 0.0005 5.1%

Mg(s) SO262 T6A23 0.0001 5.1%

Fe(s) NGA129 T6A23 0.0001 2.7%

Fe(s) NGA106 T6A23 0.0003 9.6%

Fe(s) CIW8 GH.58E-

Col

0.0010 2.4%

Table 8. Overview of the QTL3environment interactions identified

in the Ler/Kond and Ler/An-1 populations

Traits are seed mineral (Zn, Mn, K, Fe, Ca, Mg, and P) concen-trations. For the QTLs, the chromosome number (Chrom.), theirphysical position (based on the Columbia genome; in Mb) and theirclosest genetic marker (Locus) is provided, as is the probability (P)value of the interaction. For co-locating QTLs, of which only one QTLshowed interaction, the P-value of the other QTL is indicated as non-detected (nd).6

Trait Chrom Position Locus P-value

Mn 1 3.1 SNP107 P <0.001

K 1 3.1 SNP107 Nd

Fe 1 11.4 SNP373 P <0.001

Ca 1 20.6 nga128 P <0.001

Mg 1 26 SNP177 P <0.001

Zn 1 29.8 SNP142 P <0.001

P 2 7.3 F12A24b P <0.012

Zn 2 11.2 Erecta P <0.004

Mn P <0.001

Fe P <0.001

K P <0.001

Mg P <0.001

P P <0.039

Zn 3 0.8 nga172 P <0.012

Mn P <0.001

Fe P <0.001

K P <0.011

Mg P <0.001

P nd

IP6 P <0.001

Ca 3 5.8 SNP81 P <0.001

Mg 3 5.8 SNP81 P <0.001

Zn 3 8.2 SNP35 nd

P 3 19.6 SNP188 nd

Fe 3 19.6 SNP188 P <0.001

K 3 21.9 SNP197 nd

Fe 4 0.3 FRI P <0.001

Ca 4 0.3 FRI P <0.001

Mg 4 5.3 SNP117 P <0.001

Mn 4 7.8 SNP295 P <0.001

K 4 7.8 SNP295 nd

P 4 8.9 SNP199 nd

Zn 4 12.4 SNP334 nd

Mn 4 16.9 F8D20 P <0.001

Ca 4 17.5 SNP53 P <0.001

Mn 5 3.5 SNP77 P <0.001

Ca 5 3.5 SNP77 P <0.001

Zn 5 7.7 SNP236 P <0.001

Mn P <0.001

K P <0.023

Mg P <0.001

P P <0.001

K 5 25.5 MBK5 P <0.042

Ca P <0.001

Mg P <0.001

P P <0.003


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or on hydroponics, indicates the relevance of mapping QTLfor traits in different growth conditions and/or populations

to understand the environmental effect on mineral homeo-

stasis in plants better.

The latter is especially important when QTL mapping is

performed in crop plants. If commercial crops are grown

under certain conditions, then most relevant results will be

obtained when also mapping mineral QTLs in those crops

using populations grown under the same conditions. QTLsidentified in populations which were grown under dissimilar

growth conditions might not be used to improve the crop

value under commercial growth conditions. Despite the ease

of growing plants reproducibly on hydroponics medium in

a climate chamber, we do not advocate the use of such

growing conditions for mineral QTL analysis when trying

to identify relevant genetic loci controlling mineral accumu-

lation for plants grown on soil.

Supplementary data

Supplementary data are available at JXB online.

Supplementary Table 1. List of SSLP markers and PCR

primers used for genotyping of the Ler3Eri-1 RIL population.

Acknowledgements

We cordially thank and acknowledge Sigi Effgen for perform-

ing the AFLP analysis and Diaan Jamar for performing the

phytate analysis.

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A strong effect of growth medium and organ type on the identification

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